EP2105727B1 - Scanning electron microscope comprising a film for holding a sample and a dish for receiving sample material from a damaged film - Google Patents
Scanning electron microscope comprising a film for holding a sample and a dish for receiving sample material from a damaged film Download PDFInfo
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- EP2105727B1 EP2105727B1 EP09250792.0A EP09250792A EP2105727B1 EP 2105727 B1 EP2105727 B1 EP 2105727B1 EP 09250792 A EP09250792 A EP 09250792A EP 2105727 B1 EP2105727 B1 EP 2105727B1
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- sample
- film
- primary beam
- vacuum chamber
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
- G01N23/225—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion
- G01N23/2251—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion using incident electron beams, e.g. scanning electron microscopy [SEM]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
- G01N23/2204—Specimen supports therefor; Sample conveying means therefore
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/02—Details
- H01J37/20—Means for supporting or positioning the objects or the material; Means for adjusting diaphragms or lenses associated with the support
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/02—Details
- H01J37/22—Optical or photographic arrangements associated with the tube
- H01J37/226—Optical arrangements for illuminating the object; optical arrangements for collecting light from the object
- H01J37/228—Optical arrangements for illuminating the object; optical arrangements for collecting light from the object whereby illumination and light collection take place in the same area of the discharge
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/26—Electron or ion microscopes; Electron or ion diffraction tubes
- H01J37/261—Details
- H01J37/265—Controlling the tube; circuit arrangements adapted to a particular application not otherwise provided, e.g. bright-field-dark-field illumination
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/26—Electron or ion microscopes; Electron or ion diffraction tubes
- H01J37/28—Electron or ion microscopes; Electron or ion diffraction tubes with scanning beams
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2223/00—Investigating materials by wave or particle radiation
- G01N2223/30—Accessories, mechanical or electrical features
- G01N2223/307—Accessories, mechanical or electrical features cuvettes-sample holders
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2223/00—Investigating materials by wave or particle radiation
- G01N2223/60—Specific applications or type of materials
- G01N2223/612—Specific applications or type of materials biological material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2223/00—Investigating materials by wave or particle radiation
- G01N2223/60—Specific applications or type of materials
- G01N2223/637—Specific applications or type of materials liquid
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
- H01J2237/18—Vacuum control means
- H01J2237/182—Obtaining or maintaining desired pressure
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
- H01J2237/20—Positioning, supporting, modifying or maintaining the physical state of objects being observed or treated
- H01J2237/2002—Controlling environment of sample
- H01J2237/2003—Environmental cells
- H01J2237/2004—Biological samples
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
- H01J2237/244—Detection characterized by the detecting means
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
- H01J2237/26—Electron or ion microscopes
- H01J2237/28—Scanning microscopes
- H01J2237/2803—Scanning microscopes characterised by the imaging method
- H01J2237/2808—Cathodoluminescence
Definitions
- the present invention relates to an apparatus and method capable of well observing or inspecting a sample consisting of a liquid sample or cultured biological cells.
- Living organisms including we human beings are multicellular animals. Living organisms develop diseases if information cannot be transmitted normally among cells or if viruses or chemical substances cling to cells. For this reason, in the fields of molecular biology and pharmaceutics, research is conducted by peeling off cells from a living organism, cultivating the cells on a laboratory dish, giving a stimulus such as electricity, chemical substance, or medicine to the cells, and observing the resulting reaction on the cellular level.
- a sample to be inspected with an inspection apparatus incorporating SEM capabilities is normally placed in a sample chamber whose internal pressure has been reduced by vacuum pumping.
- the sample placed in the sample chamber which in turn is placed in a reduced-pressure ambient in this way, is irradiated with an electron beam (charged particle beam).
- Secondary signals such as secondary electrons or backscattered electrons produced from the sample in response to the irradiation are detected.
- One conceivable method of inspecting a sample using SEM without exposing the sample to a reduced-pressure ambient in this way consists of preparing a sample holder (sample capsule that may or may not be hermetically sealed) whose opening (aperture) has been sealed off by a film, placing the sample in the holder, and installing the holder in an SEM sample chamber that is placed in the reduced-pressure ambient.
- sample capsule The inside of the sample holder in which the sample is placed is not evacuated.
- the film that covers the opening formed in the sample holder (sample capsule) can withstand the pressure difference between the reduced-pressure ambient inside the SEM sample chamber and the ambient (e.g., atmospheric-pressure ambient) of the inside of the sample holder that is not pumped down. Furthermore, the film permits an electron beam to pass therethrough (see patent document 1).
- a culture medium is first put into a sample capsule together with cells.
- the cells are cultivated on the film.
- the sample capsule is placed into an SEM sample chamber that is in a reduced-pressure ambient.
- An electron beam is directed at the sample placed within the sample capsule from outside the capsule via the film on the capsule.
- Backscattered electrons are produced from the irradiated sample.
- the backscattered electrons pass through the film on the capsule and are detected by a backscattered electron detector mounted in the SEM sample chamber. Consequently, an SEM image is derived.
- the sample is sealed in the closed space and so it has been impossible to give a stimulus to cells or to manipulate them using a manipulator or pipette.
- the amount of the culture medium put into the sample capsule is about 15 ⁇ l. Therefore, as the culture medium evaporates, the salinity concentration rises, making it difficult to culture cells. Where the cells should be observed or inspected in vivo, there arises a problem.
- This problem can be solved by increasing the size of the sample capsule to increase the capacity.
- the film is damaged either by a stimulation induced by an electron beam or by a mechanical stimulus, a new problem is created. That is, the inside of the apparatus is contaminated with a large amount of culture medium.
- patent document 2 Examples in which two films of the structure described above are placed opposite to each other with a sample interposed between the films and in which an image is acquired by a transmission electron microscope are described in patent documents 2 and 3. Especially, patent document 2 also states a case in which an SEM image of the sample interposed between such films is acquired.
- Patent document 4 discloses a sample inspection apparatus equipped with an open-close valve for partitioning the space between a film and primary beam irradiation means within a vacuum chamber in order to permit the sample held on the film to be exchanged quickly and to prevent contamination of the inside of the vacuum chamber.
- the resolution of an optical microscope is not high enough to observe very tiny regions of biological cells. Imaging using SEM is required.
- a sample (cells) cultured on a laboratory dish is sealed into a sample capsule.
- the sample is irradiated with an electron beam via the film formed on the sample capsule.
- the sample is imaged.
- the sample capsule is a narrow closed space. Therefore, there is the problem that it has been impossible to directly observe the state of the sample immediately after a stimulus is given from the outside to the sample using a manipulator or pipette. Furthermore, the capacity inside the sample capsule is small. Consequently, when moisture evaporates and the salinity concentration rises, it is difficult to culture cells for a long time inside the sample capsule. Hence, there are problems in observing cells for a long time.
- the present invention is intended to provide an apparatus and method for inspecting a sample in such a way that biological cells held in a liquid state can be manipulated from the outside with a manipulator, pipette, or the like and that consideration is given to long-term observation.
- Patent document 4 states that when a sample is exchanged, the space between the film and the primary beam irradiation means is partitioned off by the open-close valve and that under this condition, only the space on the film side is returned to the normal pressure. It also states that if the film is damaged during inspection of the sample, the valve is closed, partitioning off the space inside the vacuum chamber to thereby prevent contamination into the vacuum chamber.
- the present invention relates to a sample inspection apparatus and method free of these problems. It would be desirable to provide a sample inspection apparatus that can be easily maintained and serviced by certainly preventing at least the inside of primary beam irradiation means from being contaminated. It would be further desirable to provide a sample inspection-method implemented by the sample inspection apparatus.
- a sample inspection apparatus has a film including a first surface to hold a sample thereon, a vacuum chamber for reducing the pressure of an ambient in contact with a second surface of the film, primary beam irradiation means which is connected with the vacuum chamber for irradiating the sample with a primary beam via the film, signal detection means for detecting a secondary signal produced from the sample in response to the beam irradiation, a partitioning member , having a receiver dish, located in the vacuum chamber configured to move between two extreme positions, one extreme position in an open state and the other extreme position in a closed state, in the open state the partitioning member does not interrupt an optical axis of the primary beam, in the closed state the partitioning member is moved to a position between the film and the primary beam irradiation means which are located opposite to each other and interrupts an optical axis of the primary beam without hermetically isolating a side on which the film is located from a side on which the primary beam irradiation means is located within the vacuum chamber
- detection means which, if there is any damage to the film, can detect the damage.
- the partitioning member is configured to move to the closed state in a-position between the film and the primary beam irradiation means so that the partitioning member interrupts the optical axis of the primary beam.
- the inside of the primary beam irradiation means and the inside of the vacuum chamber can be returned to the atmospheric pressure. In this case, gas can be supplied into the vacuum chamber via the inside of the primary beam irradiation means.
- the partitioning plate may have a receiver dish structure capable of receiving and stopping the sample flowing into the vacuum chamber when the film is damaged.
- the first surface may hold the sample in an open state in which access to the surface from the outside is allowed.
- a manipulator having a front-end portion capable of being brought close to or making contact with the sample and optical image acquisition means for observing the sample and the manipulator may be provided.
- the partitioning plate is automatically operated in response to the detection.
- the detection means detects a rise in pressure inside the vacuum chamber caused by the damage to the film.
- the first surface of the film may be the upper surface of the film.
- the second surface of the film may be the lower surface of the film.
- the primary beam is a beam of charged particles or an electron beam.
- the secondary signal can be at least one type of secondary electrons, backscattered electrons, X-rays, and cathodoluminescent light.
- An inspection method starts with holding a sample on a first surface of a film.
- the pressure of a space inside the vacuum chamber in contact with a second surface of the film is reduced.
- the sample is irradiated with a primary beam via the film by primary beam irradiation means.
- a secondary signal produced from the sample in response to the beam irradiation is detected to inspect the sample.
- This method is characterized in that when any damage to the film is detected, a partitioning member having a receiver dish interrupts an optical axis of the primary beam by moving to a position between the film and the primary beam irradiation means without hermetically isolating a side on which the film is located from a side on which the primary beam irradiation means is located within the space in order that the receiver dish receives the sample that flows into the vacuum chamber when the film is broken.
- the partitioning plate when any damage to the film is detected, the partitioning plate can partition off the region. Concurrently, the inside of the primary beam irradiation means and the inside of the vacuum chamber can be returned to the atmospheric pressure. In this case, gas can be supplied into the vacuum chamber via the inside of the primary beam irradiation means.
- the partitioning plate may have a receiver dish structure capable of stopping and accepting the sample.
- the first surface may hold the sample in an open state in which access to the surface from the outside is allowed.
- the sample may be manipulated.
- an optical image of the sample may be acquired.
- the damage to the film can be detected based on a rise in pressure in the vacuum chamber caused by the damage to the film.
- the first surface of the film may be the upper surface of the film.
- the second surface of the film may be the lower surface of the film.
- the primary beam is a beam of charged particles or an electron beam.
- the secondary signal can be at least one type of secondary electrons, backscattered electrons, X-rays, and cathodoluminescent light.
- the partitioning plate partitions off the region across which the film and the primary beam irradiation means are located opposite to each other without hermetically isolating the side on which the film is located from the side on which the primary beam irradiation means is located within the vacuum chamber.
- the partitioning plate partitions off the region across which the film and the primary beam irradiation means are located opposite to each other without hermetically isolating the side on which the film is located from the side on which the primary beam irradiation means is located in the space in contact with the second surface of the film.
- the partitioning plate quickly partitions off the region across which the film and primary beam irradiation means are located opposite to each other. Hence, contamination of at least the primary electron beam irradiation means can be prevented with certainty.
- the sample inspection apparatus can be maintained and serviced easily. Additionally, damage to the primary electron beam irradiation means, which is expensive, can be prevented.
- Fig. 1 is a schematic diagram of a first embodiment of the sample inspection apparatus according to the present invention.
- the apparatus consists chiefly of an optical microscope 27, a manipulator 26, and an electron beam apparatus section 29 located under a sample holder 40.
- the electron beam apparatus section 29 includes an electron optical column 1 forming primary beam irradiation means.
- An electron gun 2 forming an electron source is disposed in the electron optical column 1 and emits an accelerated electron beam 7 that is a primary beam.
- the electron beam 7 is one kind of charged particle beam.
- the beam 7 is focused by a condenser lens (objective lens) 3.
- the focused electron beam 7 is directed at a liquid sample 20 via a sample-holding film 32 (described later) formed on the sample holder 40.
- the sample 20 is held on the sample holder 40.
- the liquid sample 20 includes biological cells and a culture medium.
- the upper side of the sample 20 is open and in contact with a normal-pressure (atmospheric-pressure) ambient.
- the front-end side of the electron optical column 1 is connected with a vacuum chamber 11.
- the electron gun 2 is mounted in the base side of the optical column 1.
- the base side of the column 1 is located under the vacuum chamber 11. Because of this configuration, the electron beam 7 released from the electron gun 2 travels upward through the optical column 1, passes through the space inside the vacuum chamber 11 and through the sample-holding film 32, and reaches the liquid sample 20.
- the electron beam 7 is deflected by deflection means (not shown).
- the beam 7 scans the liquid sample 20.
- a specimen contained in the liquid sample 20 is also scanned with the beam 7.
- the electron optical column 1 forms the primary beam irradiation means in this way.
- the column is of the inverted type.
- a backscattered electron detector 4 is mounted on the front-end side of the optical column 1 inside the vacuum chamber 11.
- the backscattered electron detector 4 detects backscattered electrons produced when the specimen included in the liquid sample 20 is irradiated with the electron beam 7.
- a semiconductor detector using a PN junction or a scintillator detector using a YAG crystal is used as the backscattered electron detector 4.
- the output signal from the backscattered electron detector 4 is sent to an image formation device 22 disposed outside the vacuum chamber 11.
- the image formation device 22 forms image data based on the output signal from the detector 4.
- the image data corresponds to an SEM image, and is sent to a display device 23.
- the display device 23 displays an image based on the image data sent in.
- the displayed image forms an SEM image.
- the image data formed by the image formation device 22 is sent to a computer 25.
- the computer 25 image-processes the image data and makes decisions based on the result of the image processing.
- the inside of the electron optical column 1 is pumped down to a desired pressure by vacuum pumping means 8.
- the inside of the vacuum chamber 11 is evacuated to a desired pressure by vacuum pumping means 9.
- the vacuum chamber 11 is placed over a pedestal 10 via a vibration-proofing device 13.
- a sample holder placement portion 12 is formed on top of the vacuum chamber 11 and provided with a hole to permit the electron beam 7 to be directed at the sample-holding film 32.
- the sample holder 40 is placed on the placement portion 12 via an O-ring (not shown). Consequently, the sample holder 40 is withdrawably supported in the vacuum chamber 11.
- a vacuum gauge 15 for detecting the pressure inside the vacuum chamber 11 is mounted in the vacuum chamber 11.
- a partitioning plate or partitioning member 14 is mounted between the front end of the electron optical column 1 and the sample-holding film 32. In Fig. 1 , the partitioning plate 14 is in its open state. If the film 32 is damaged and the vacuum gauge 15 has detected a given rise in the pressure, the partitioning plate 14 automatically moves as shown in Fig. 2 . For example, if the film 32 is damaged, the liquid sample 20 flows into the vacuum chamber 11, and the pressure inside the vacuum chamber increases above 100 Pa, then the partitioning plate 14 is moved as shown in Fig. 2 .
- the partitioning plate 14 partitions off the region across which the sample-holding film 32 and the electron optical column 1 are disposed opposite to each other. Inside the sample chamber, the side on which the sample-holding film 32 is located is not hermetically isolated from the side on which the electron optical column 1 is located.
- the liquid sample 20 entered in the vacuum chamber 11 can be received and stopped by the partitioning plate 20, thus preventing contamination of the electron optical column 1 and backscattered electron detector 4. If the partitioning plate 14 is equipped with a receiver dish that receives liquid sample, then it is possible to cope with inflow of a large amount of liquid sample.
- the partitioning plate 14 vertically partitions the inside of the vacuum chamber 11 into two spaces not completely but partially in this way. Therefore, the structure is simple.
- the partitioning plate can be operated at high speed.
- the partitioning plate can be set into operation (brought to the state of Fig. 2 from the state of Fig. 1 ) after a lapse of 0.1 second since the vacuum gauge 15 detected a rise in pressure.
- the partitioning plate can be thinned. Consequently, the distance between the front end of the electron optical column 1 and the sample-holding film 32 (i.e., the working distance of the SEM) can be reduced, thus achieving high resolution.
- the electron beam apparatus section 29 having the electron optical column 1, vacuum chamber 11, partitioning plate 14, and vacuum gauge 15 is controlled by an electron beam controller 24.
- the manipulator 26 for giving a stimulus (such as a voltage, chemical substance, or medicine) to the sample and for moving it if necessary and an optical microscope 27 are placed on the sample holder placement portion 12.
- the microscope 27 permits one to observe the sample and to check the position of the manipulator 26.
- the optical axis of the optical microscope 27 is coincident with the optical axis of the electron beam 7.
- the center of field of view of the optical microscope 27 is coincident with the center of field of view of the SEM image.
- a region observed by the optical microscope can be made substantially coincident with the SEM image.
- the field of view of the SEM image and the field of view of the optical microscope 27 can be adjusted by manipulating the manipulator 26 ormoving the sample holder placement portion 12 on which the sample holder 40 is placed by means of a moving mechanism (not shown).
- the inspection apparatus has the electron beam apparatus section 29, manipulator 26, optical microscope 27, electron beam controller 24, overall controller 28, image formation device 22, and display device 23. These portions are connected with the computer 25. Information can be exchanged between these portions.
- the sample holder 40 is constructed as shown in Fig. 3 .
- the sample holder 40 is composed of a dish-like body portion 37 made of plastic or glass and a film holder (framelike member) 18 on which the sample-holding film 32 is formed.
- the film 32 transmits the electron beam 7.
- a recessed portion is formed inside the body portion 37.
- the bottom surface of the recessed portion forms a sample-holding surface 37a that is open.
- the sample-holding surface 37a of the body portion 37 is (centrally in the example of Fig. 3 ) provided with a through-hole 37b.
- a step portion 37c is formed around the hole 37b on the side of the sample-holding surface 37a.
- the film holder 18 is disposed on the step portion 37c and has the sample-holding film 32.
- the sample-holding film 32 has a first surface 32a that forms the sample-holding surface 37a.
- the sample-holding surface 37a is substantially flush with the sample-holding surface 37a of the body portion 37. Consequently, at least a part of the sample-holding surface 37a of the sample holder 40 is formed by the sample-holding film 32.
- Tapering portions 37d are formed on the side of the hole 37b on the opposite side of the sample-holding surface 37a.
- the tapering portions 37d are spread apart toward the surface on the opposite side of the sample-holding surface 37a.
- the spread angle is set to 90° to 120°.
- a region of the lower surface of the sample holder 40 might be exposed to a vacuum ambient and become irradiated with the electron beam 7.
- a conductive film 301 is formed on this region to prevent charging of the sample holder 40 when it is irradiated with the beam 7.
- the conductive film 301 is in contact with the film holder 18 . Electric charge accumulated by being irradiated by the electron beam 7 can be dissipated away to the liquid sample 20 via the film holder 18 made of silicon.
- the presence of the conductive film 301 reduces the charging of the lower surface of the sample holder 40 and can prevent displacement of the orbit of the beam 7 (that would normally be produced when the liquid sample 20 is illuminated with the beam 7) and distortion and illumination spots in the SEM image that would be normally produced by displacement of the orbit of backscattered electrons.
- the conductive film 301 can be formed, for example, by vapor-depositing aluminum or gold or applying silver paste.
- the structure of the film holder 18 is shown in Fig. 4 .
- the sample-holding film 32 is formed on a silicon substrate 34.
- the first surface 32a of the sample-holding film 32 (lower surface as viewed in Fig. 4 ; upper surface as viewed in Fig. 3 ) is exposed.
- the liquid sample 20 containing liquid such as a culture medium and sample cells is placed on the first surface (sample-holding surface) 32a of the sample-holding film 32. Since the first surface 32a is under atmospheric pressure, evaporation of moisture from the liquid sample 20 can be suppressed to a minimum.
- the silicon substrate 34 is centrally provided with an opening 34a (upper surface in Fig. 4 ; lower surface in Figs. 1 and 3 ) covered with the sample-holding film 32.
- a central portion of the second surface 32b of the sample-holding film 32 is exposed to the inside ambient of the vacuum chamber 11 through the opening 34a.
- the first surface 32a of the sample-holding film 32 is exposed to the atmospheric-pressure ambient, while the second surface 32b is exposed to the vacuum ambient.
- the film 32 is supported and reinforced with a lattice 35.
- Fig. 5(a) silicon nitride, films 502 and 503 are formed on a silicon substrate 501 using CVD (chemical vapor deposition). A typical thickness of the films 502 and 503 is 30 nm. Layers of resist 504 and 505 are applied on the silicon nitride films 502 and 503, respectively ( Fig. 5(b) ). The layer of resist 505 is patterned photolithographically to leave behind resist layer portions 505a ( Fig. 5(c) ). Using the resist pattern as a mask, the silicon nitride film 503 is processed by dry etching, and silicon nitride film portions 503a are left behind ( Fig. 5(d) ).
- CVD chemical vapor deposition
- the silicon substrate 501 is wet-etched with KOH to form an opening 510 ( Fig. 5(e) ).
- the resist layer portions 504 and 505a are removed by ashing ( Fig. 5(f) ).
- the film holder 18 is completed at this point.
- Resist 506 is applied on the layer of silicon nitride film 502 ( Fig. 5(g) ).
- a layer of metal 507 of A1 or Ni is formed to a thickness of 1 ⁇ m on the opposite side of the silicon nitride film 502 ( Fig. 5(h) ).
- Resist 508 is applied on the metal layer 507, and a pattern is photolithographically formed using a mask ( Fig. 5 (i) ). Using the resist layer 508 as a mask, the metal layer 507 is etched ( Fig. 5(j) ). Finally, the resist layer 508 is removed by ashing or organic cleaning ( Fig. 5(k) ). As a result, the opening 34a and lattice 35 are formed.
- the film holder 18 fabricated in this way is inverted up and down from the state of Fig. 4 .
- the first surface 32a of the silicon nitride film 502 that is the sample-holding film 32 is taken as an upper surface.
- the second surface 32b can also be taken as an upper surface.
- the film holder 18 is firmly attached to the step portion 37c over the hole 37b formed in the body portion 37 forming the sample holder 40.
- the sample holder 40 is fabricated ( Fig. 3 ).
- bonding using an epoxy-based or silicone-based adhesive or fusion making use of heat, ultrasonic waves, or laser light can be used. Consequently, the film holder 18 is firmly held in a position corresponding to the hole 37b in the sample-holding surface 37a of the body portion 37.
- the body portion 37 and film holder 18 are combined to fabricate the sample holder 40.
- the sample-holding film 32 may be directly firmly bonded to the body portion 37.
- the body portion 37 and the sample-holding film 32 may be fabricated integrally.
- cell adhesion molecules acting as molecules for bonding the sample may be applied to the sample-holding surface 37a including the first surface 32a of the sample-holding film 32.
- the thickness of the silicon nitride film 502 is set to a range of from 10 to 1,000 nm.
- the sample-holding film 32 of the film holder 18 is made of silicon nitride.
- the film 32 may bemade of silicon oxide, boron nitride, polymer, polyethylene, polyimide, polypropylene, orcarbon. Where films of these materials are used, their film thicknesses are set to a range of from 10 to 1,000 nm.
- the sample-holding film 32 made of the aforementioned material transmits the electron beam 7 but does not transmit gas or liquid. Moreover, it is necessary that the film be capable of withstanding a pressure difference of at least 1 atmosphere across the opposite surfaces.
- the preferable thickness of the film is 20 to 200 nm.
- FIG. 3 A sample inspection method according to the present invention is next described.
- cells 38 forming a sample are cultured within a culture medium 39 using the sample holder 40.
- a normal method as described below is used.
- the culture medium is Product No. D5796 of Sigma-Aldrich Co., for example.
- the culture medium is discarded from the laboratory dish where the cells have been previously cultured.
- a mixture liquid of trypsin and EDTA (ethylenediaminetetraacetic acid) is put into the dish to peel off the cells adsorbed to the dish.
- the peeled cells are recovered into a centrifuge tube.
- a culture medium is put into the tube.
- the trypsin is inactivated and then the cells are spun down.
- the supernatant fluid is discarded from the centrifuge tube and the remaining liquid is stirred in the culture medium.
- Apart (e.g., 1/10) of the stirred liquid including the cells 38 is entered into the sample holder 40.
- the culture medium (liquid sample) 39 is grafted.
- the holder is allowed to stand still in a cell culture chamber.
- the cells 38 begin to be adsorbed onto the sample-holding surface 37a of the sample holder 40 including the surface 32a of the sample-holding film 32 and proliferate.
- the aforementioned method may be modified according to cells, and is merely one example. Consequently, the cells 38 which are to be observed or inspected and become a sample are cultured within the sample holder 40. It follows that the liquid sample 20 containing the cultured cells 38 and culture medium 39 is constituted.
- cell adhesion molecules molecules for bonding of the sample
- the cell adhesion molecules cause cells arranged for cultivation and cells proliferated by cultivation to be adsorbed onto the sample-holding surface.
- the cell adhesion molecules include collagen, fibronectin, vitronetin, cadherin, integrin, claudins, desmogleins, neuroligin, neurexin, selectin, laminins, and poly-L-lysine.
- the sample holder 40 After the cells are cultured within the sample holder 40 as described above, the sample holder 40 is placed on the holder placement portion 12. At this time, the partitioning plate 14 is closed and in the state of Fig. 2 . Subsequently, the insides of the vacuum chamber 11 and electron optical column 1 are evacuated to desired degrees of vacuum using the vacuum pumping means 8 and 9. For example, the pressure inside of the electron optical column 1 is set below 1 Pa. The pressure inside the electron optical column 1 (especially, around the electron gun 2) is set to about 10 -4 to 10 -5 Pa, for example.
- the positions of the cells 38 and of the manipulator 26 are then checked with the optical microscope 27.
- a glass microtube holding microelectrodes therein is installed at the front end of the manipulator.
- a voltage can be applied to the cells through the microelectrodes.
- a liquid can be made to flow in and out through the glass microtube for manipulation.
- the manipulator 26 is moved while making an observation with the optical microscope 27 to bring the cells 38 close to the glass microtube. Then, a negative pressure is applied to the glass microtube to bring it into intimate contact with the cell membranes. As a result, potential response can be measured.
- the manipulator 26 When the manipulator 26 is moved as described above, if the sample-holding film 32 is erroneously damaged or destroyed, and if the liquid sample 20 flows into the vacuum chamber 11, the sample 20 can be received and stopped by the partitioning plate 14 because the plate is closed. Consequently, the electron optical column 1 is not contaminated. Where the partitioning plate 14 is not present as in the prior art, the liquid sample 20 enters the electron optical column 1, making it necessary to clean or replace the column. As a result, the apparatus is made usable.
- the partitioning plate 14 is opened.
- the inside of the vacuum chamber 11 is ceased to be partitioned.
- the light illumination of the optical microscope 27 is ceased.
- Other extraneous light is blocked in a manner not shown. The blocking also shields the filmholder 18 and liquid sample 20 against radiation rays produced when the electron beam 7 hits the film holder 18 and sample 20.
- the electron beam 7 is directed at the liquid sample 20 including the cells 38 from the electron optical column 1 to perform imaging.
- the beam 7 passes through the sample-holding film 32 of the sample holder 40 and hits the cells 38.
- Backscattered electrons produced from the cells 38 in response to the illumination are detected by the backscattered electron detector 4.
- the aforementioned tapering portions 37d are formed around the hole 37b of the body portion 37 forming the sample holder 40, collision of the backscattered electrons against the inner side surface of the hole 37b can be suppressed to a minimum. That is, the backscattered electrons can be suppressed from being blocked. The backscattered electrons can be detected efficiently by the backscattered electron detector 4.
- a detection signal produced from the backscattered electron detector 4 is fed to the image formation device 22, which in turn forms image data based on the detection signal. Based on the image data, an image (SEM image) is displayed on the display device 23.
- an electrical stimulus is given to the cells 38 using the microelectrodes installed at the front end of the manipulator 26 to manipulate the cells.
- An SEM image is acquired in the same way as in the above-described process step. The response of the cells 38 to the stimulus is checked.
- the partitioning plate 14 is closed to prevent contamination of the electron optical column 1 if the sample-holding film 32 should be damaged. Before a variation caused by application of a stimulus to the cells 38 is observed by SEM as described above, an observation may be made with the optical microscope 27. Also, at this time, if the partitioning plate 14 is closed, risk of contamination occurring when the sample-holding film 32 is broken can be reduced.
- the probability of contamination of the inside of the apparatus can be reduced by shortening the interval for which the partitioning plate 14 is opened during inspection.
- the partitioning plate 14 may be once closed.
- the plate 14 may be again opened at a time when a reaction is deemed to have taken place.
- imaging may be performed using the electron beam 7.
- the reaction can be checked with the optical microscope 27.
- the manipulator 26 can have a mechanism capable of spraying a chemical substance or medicine into the liquid sample 20. Behavior of the cells 38 in response to the chemical substance or medicine can be observed or inspected while observing the cells by SEM. Furthermore, a function of permitting a liquid to flow out can be imparted to the manipulator 26. This permits the sprayed substance to be recovered. Also, the pH of the culture medium and the osmotic pressure can be maintained constant.
- backscattered electrons are used to form an image.
- Backscattered electrons produce a signal intensity proportional to the atomic number. Therefore, where the sample is almost totally made of substances of low atomic numbers such as a biological sample, the image contrast is very low, and it is difficult to improve the resolution.
- a heavy metal such as gold may be adsorbed onto portions of the cells 38 to be noticed in their behavior.
- gold is adsorbed onto the portions (antigen) via an antibody by causing the antigen tagged with gold particles having the nature of being adsorbed on the portions (antigen) to be sprayed against the cells by making use of an antigen-antibody reaction.
- a fluorescent dye or quantum dots e.g., nanoparticles of Si or particles of CdSe coated with ZnS and having sizes of 10 to 20 nm
- emit light when irradiated with an electron beam may be previously adsorbed onto certain portions of the cells 38, and the emitted light may be observed with an optical microscope.
- normally used gold particles have particle diameters of 10 to 30 nm.
- the adsorptive force between the antibody and gold particles is weak, and gold particles of 10 to 30 nm may not be attached.
- very small gold particles nanogold particles having particle diameters of the order of nanometers are first attached to the antibody. Under this condition, the gold particles are too small and it is difficult to observe them by SEM.
- Silver is adsorbed around the gold particles by making use of a silver sensitizer. This makes it easier to detect them by SEM.
- cells previously cultured in a laboratory dish are taken out and grafted onto the sample holder 40. Then, the cells are cultured.
- cells may be taken from a living organism and directly placed on the sample-holding surface 37a of the sample holder 40. The cells may be cultured in the sample holder 40.
- the present invention makes it possible to observe a specimen by SEM via the sample-holding film 32, the specimen being included in a liquid.
- the use of an open sample chamber facilitates giving a stimulus to (or, manipulating) cells using the manipulator because access to the sample can be made from the outside.
- the partitioning plate 14 is moved as shown in Fig. 2 .
- the liquid sample 20 is received and stopped by the partitioning plate 14.
- the amount of the liquid sample 20 is large, it is possible to prepare a receiver dish on the partitioning plate 14. It takes only 0.1 second until the partitioning plate 14 is closed after detection of a pressure rise.
- the contamination of the electron optical column 1 can be reduced to a level at which no cleaning is necessary. In this way, the present invention enhances convenience in use of the apparatus.
- the amount of culture medium can be increased. Cells are allowed to survive for a long time. SEM imaging can be performed for a long time.
- the inverted type SEM is used.
- a normal non-inverted type SEM using a sealed sample capsule as described in the "Description of the Related Art" of this specification can be used without problem.
- the partitioning plate 14 needs to be located between the sealed sample capsule and the front end of the electron optical column 1.
- an electron beam is used as the primary beam. If the sample-holding film 32 shows sufficient shock resistance and strength against impingement of other charged particle beam such as a helium ion beam, the invention can also be applied in a case where the other charged particle beam is used.
- backscattered electrons are used as a secondary signal.
- Information about the cells 38 can also be obtained by detecting other form of information such as secondary electrons, X-rays, cathodoluminescent light, and electric current absorbed into the cells 38 forming a sample. It is convenient to use the manipulator 26 in measuring the absorption current.
- the sample-holding film 32 of the present embodiment withstand a pressure difference of at least 1 atm. and that gas or liquid do not flow in or out.
- the material of the film 32 includes at least one of polymer, polyethylene, polyimide, polypropylene, carbon, silicon oxide, silicon nitride, and boron nitride.
- the partitioning plate 14 and backscattered electron detector 4 are fabricated separately. As a modified embodiment, they may be integrated as shown in Fig. 6 . That is, in the embodiment of Fig. 6 , the backscattered electron detector 4 is mounted at the front end of the partitioning plate 14. In this structure, when the partitioning plate 14 is opened, the backscattered electron detector 4 is located immediately above the electron optical column 1, maximizing the efficiency at which backscattered electrons are detected.
- FIG. 7 A second embodiment of the sample inspection apparatus of the present invention is next described by referring to Fig. 7 .
- the sample inspection apparatus shown in Fig. 7 inspects a sample by irradiating cells 110 held on a sample-holding film 150 with an electron beam from below via the film 150.
- the sample-holding film 150 is held in a laboratory dish 130.
- a sample 140 consisting of a liquid culture medium and the cells 110 is held in the dish 130.
- the dish 130 is placed over a vacuum chamber 230 via an O-ring 160.
- An opening is formed in an upper part of the vacuum chamber 230 and located opposite to the sample-holding film 150 in the laboratory dish 130.
- the front end of an electron optical column 200 acting as primary beam irradiation means is connected with a lower part of the vacuum chamber 230.
- the column 200 is controlled by an SEM control circuit 205.
- an electron beam is emitted from an electron gun 2500 disposed in the electron optical column 200 toward the sample 140.
- backscattered electrons are produced from the cells 110 and detected by a backscattered electron detector 210.
- the output signal from the detector 210 based on the detected backscattered electrons is amplified by a preamplifier 215 and accepted into a control computer 1300 via the SEM control circuit 205.
- a two-dimensional image based on a signal from the control computer 1300 is displayed as a sample image on a display monitor 1400.
- An X-ray shielding cover 250 is mounted to prevent X-rays produced from the vicinities of the sample in response to the electron beam irradiation from leaking from the apparatus.
- the ambients inside and outside the cover 250 are at normal pressure (atmospheric pressure).
- the inside of the vacuum chamber 230 is evacuated via a tube 231 by a vacuum pump 2100.
- the tube 231 is connected with the bottom surface of the inside of the vacuum chamber 230 as shown.
- the pump 2100 is connected with the tube 231 via a valve 2105.
- a pressure gauge 2010 acting as pressure detection means is connected with the tube 231.
- a leaking gas supply source 2220 is connected also with the tube 231 via another valve 2210.
- the pressure gauge 2010 can detect the pressure inside the vacuum chamber 230 via the tube 231.
- the pressure gauge 2010, vacuum pump 2100, and valves 2105, 2210 are controlled in operation by a vacuum control system 2000.
- the inside of the electron optical column 200 is evacuated by a vacuum pump 2504 via a tube 232.
- the tube 232 is connected into the column 200 as shown.
- the pump 2504 is connected with the tube 232 via a valve 2503.
- a leaking gas supply source 2502 is connected with the tube 232 via a valve 2501.
- the vacuum pump 2504 and valves 2501, 2503 are also controlled in operation by the vacuum control system 2000.
- the backscattered electron detector 210 is mounted at the front end of the partitioning plate or partitioning member 260.
- the partitioning plate 260 is driven by a partitioning plate drive means 265, which in turn is controlled in operation by the vacuum control system 2000.
- the detector 210 is provided with an opening to permit passage of the electron beam coming from the electron optical column 200.
- the insides of the vacuum chamber 230 and electron optical column 200 are pumped down to set degrees of vacuum.
- the vacuum control system 2000 closes the valve 2210 and opens the valve 2105.
- the inside of the vacuum chamber 230 is evacuated by the vacuum pump 2100 via the tube 231.
- the vacuum control system 2000 closes the valve 2501 and opens the valve 2503. Consequently, the inside of the electron optical column 200 is evacuated by the vacuum pump 2504 via the tube 232.
- an electron beam is directed at the cells 110 forming a sample from the electron gun 2500 in the electron optical column 200 through the vacuum chamber 230.
- the beam reaches the cells 110 through the sample-holding film 150.
- Backscattered electrons produced from the cells 110 at this time pass through the sample-holding film 150 and reach the backscattered electron detector 210, where the electrons are detected. As a result, a sample image is formed.
- the sample-holding film 150 holding the sample 140 including the liquid and cells 110 is damaged or destroyed, atmosphere enters through the damaged portion of the film.
- the degree of vacuum of the ambient inside the vacuum chamber 230 deteriorates. For example, when the inside pressure is set to 1 Pa, the pressure increases rapidly to above 30 Pa. In this case, the vacuum deterioration is detected by the pressure gauge 2010, and information about it is sent to the vacuum control system 2000.
- the vacuum control system 2000 operates the partitioning plate drive means 2 65. Consequently, the partitioning plate 260 moves through the vacuum chamber 230 and is placed between the sample-holding film 150 and the electron optical column 200. At this time, the partitioning plate 260 partitions off the region across which the sample-holding film 150 and electron optical column 200 are located opposite to each other without hermetically isolating the side where the sample-holding film 150 is located from the side where the column 200 is located inside the vacuum chamber 230.
- the partitioning plate 260 can be instantly moved at high speed. If the sample-holding film 150 is damaged and the sample 140 including liquid intrudes into the vacuum chamber 230, most of the entering sample is received and stopped by the partitioning plate 260.
- the vacuum control system 2000 closes the valve 2105 on the side of the tube 231 and the valve 2503 on the side of the tube 232 and opens the valve 2501 on the side of the tube 232. Consequently, vacuum pumping through the tube 231 is stopped. Also, vacuum pumping through the tube 232 is stopped. Leaking gas such as nitrogen from the gas supply source 2502 is supplied into the electron optical column 200 via the tube 232.
- the leaking gas supplied into the electron optical column 200 reaches the inside of the vacuum chamber 230.
- the inside of the column 200 and the inside of the vacuum chamber 230 including the inside of the tube 231 are returned to the atmospheric pressure.
- the leaking gas flows into the vacuum chamber 230 from inside the column 200 via the front end of the column 200 and so if the sample-holding film 150 is damaged and the sample 140 containing liquid enters the vacuum chamber 230, the sample 140 does not enter the column 200. Consequently, contamination is prevented.
- the leaking gas can also be supplied from the gas supply source 2220 into the vacuum chamber 230 via the tube 231 by opening the valve 2210 on the side of the tube 231.
- the vacuum control system 2000 may close the valves 2501 and 2210 when the pressure gauge 2010 has detected that the pressure inside the vacuum chamber 230 has reached a pressure slightly lower than the atmospheric pressure.
- a sample inspection apparatus has a sample-holding film (32, 150) including a first surface on which a sample is held, a vacuum chamber (11, 230) for reducing the pressure of an ambient in contact with a second surface of the film, primary beam irradiation means (1, 200) connected with the vacuum chamber and irradiating the sample with a primary beam via the film, signal detection means (4, 210) for detecting a secondary signal produced from the sample in response to the beam irradiation, a partitioning member (14, 260) capable of partitioning off a region across which the film and the primary beam irradiation means (1, 200) are located opposite to each other without hermetically isolating the side on which the film is located from the side on which the primary beam irradiation means (1, 200) is located inside the vacuum chamber.
- the detection means (15, 2010) is provided which, if the film (32, 150) is damaged, detects the damage.
- the partitioning member (14, 260) can partition off the region.
- the pressure restoration means (2220, 2502) which, when the detection means (15, 2010) has detected any damage to the film, returns the inside of the primary beam irradiation means (1, 200) and the inside of the vacuum chamber (11, 230) to the atmospheric pressure.
- the pressure restoration means (2502) supplies leaking gas into the vacuum chamber (230) via the inside of the primary beam irradiation means (200), whereby the insides of the primary beam irradiation means (200) and vacuum chamber (230) can be returned to the atmospheric pressure.
- the detection means (15, 2010) can detect any damage to the film based on a rise in pressure inside the vacuum chamber (11, 230).
- the partitioning member (14, 260) can have a receiver dish structure.
- the signal detection means (4, 210) can be mounted to the partitioning member (14, 260).
- the first surface of the sample-holding surface can hold a sample under an open state to permit a manipulator to make access to the surface from the outside.
- the apparatus can have the manipulator whose front end can be brought close to or make contact with the sample held on the first surface of the film and optical image acquisitionmeans for observing the sample and the manipulator.
- the first surface of the sample-holding film can be taken as the upper surface of the film, while the second surface of the film can be taken as the lower surface of the film.
- the primary beam released from the primary beam irradiation means (1, 200) can be a beam of charged particles or electron beam.
- the secondary signal can be at least one type of secondary electrons, backscattered electrons, X-rays, and cathodoluminescent light.
- the sample inspection method according to the present invention can be implemented by inspecting a sample with the above-described sample inspection apparatus.
- the sample inspection method starts with holding a sample on a first surface of a sample-holding film (32, 150). The pressure of a space in contact with a second surface of the film is reduced. The sample is irradiated with a primary beam via the film by primary beam irradiation means (1, 200). A secondary signal produced from the sample in response to the primary beam irradiation is detected, thus inspecting the sample.
- the region across which the film and the primary beam irradiation means (1, 200) are located opposite to each other can be partitioned off by the partitioning member (14, 260) without hermetically isolating the side on which, the film is located from the side on which the primary beam irradiation means (1, 200) is located inside the space.
- the inside of the primary beam irradiation means (1, 200) and the space can be returned to the atmospheric pressure.
- the inside of the primary beam irradiation means (1, 200) and the space can be returned to the atmospheric pressure by supplying leaking gas into the space via the inside of the primary beam irradiation means (1, 200).
- the damage to the film can be detected based on a rise in pressure inside the space.
- the sample held on the first surface of the film can be manipulated.
- An optical image of the sample can be acquired.
- FIG. 8 a modification of the second embodiment is shown in Fig. 8 .
- the differences between the sample inspection apparatus shown in Fig. 8 and the apparatus shown in Fig. 7 are that the lower surface of the partitioning plate 260 is located slightly below the lower surface of the partitioning plate shown in Fig. 7 and that an 0-ring 161 is mounted to the lower surface.
- the partitioning plate-driving means 265 when the partitioning plate-driving means 265 operates, the partitioning plate 260 closes the front end of the electron optical column 200 via the O-ring 161. Consequently, the inside of the column 200 is hermetically isolated from the inside of the vacuum chamber 230.
Description
- The present invention relates to an apparatus and method capable of well observing or inspecting a sample consisting of a liquid sample or cultured biological cells.
- Living organisms including we human beings are multicellular animals. Living organisms develop diseases if information cannot be transmitted normally among cells or if viruses or chemical substances cling to cells. For this reason, in the fields of molecular biology and pharmaceutics, research is conducted by peeling off cells from a living organism, cultivating the cells on a laboratory dish, giving a stimulus such as electricity, chemical substance, or medicine to the cells, and observing the resulting reaction on the cellular level.
- In the past, optical microscopes have been used for such observation. Manipulators or pipettes have been employed to give stimuli to cells. Frequently, important portions to be observed are very tiny regions of less than 0.1 µm that are impossible to observe with an optical microscope. For example, diseases arising from inability to exchange substances normally among biological cells include hypertension, diabetes insipidus, arrhythmia, myopathy, diabetes, and deprementia. Exchange of substances among cells is performed by ion channels having sizes of about 10 nm and existing in cell membranes. Because it is difficult to observe such ion channels with optical microscopes, there has been a demand for a technique enabling observation using a scanning electron microscope (SEM) having high resolution.
- However, a sample to be inspected with an inspection apparatus incorporating SEM capabilities is normally placed in a sample chamber whose internal pressure has been reduced by vacuum pumping. The sample placed in the sample chamber, which in turn is placed in a reduced-pressure ambient in this way, is irradiated with an electron beam (charged particle beam). Secondary signals such as secondary electrons or backscattered electrons produced from the sample in response to the irradiation are detected.
- In such inspection of a sample using SEM, the sample is exposed to a reduced-pressure ambient. Therefore, moisture evaporates from the sample, so that the cells die. It has been impossible to observe reactions of living cells to a stimulus.
- Accordingly, when an inspection is performed under the condition where the sample contains moisture, it is necessary to prevent the sample from being exposed to the reduced-pressure ambient; otherwise, moisture would evaporate from the sample. One conceivable method of inspecting a sample using SEM without exposing the sample to a reduced-pressure ambient in this way consists of preparing a sample holder (sample capsule that may or may not be hermetically sealed) whose opening (aperture) has been sealed off by a film, placing the sample in the holder, and installing the holder in an SEM sample chamber that is placed in the reduced-pressure ambient.
- The inside of the sample holder in which the sample is placed is not evacuated. The film that covers the opening formed in the sample holder (sample capsule) can withstand the pressure difference between the reduced-pressure ambient inside the SEM sample chamber and the ambient (e.g., atmospheric-pressure ambient) of the inside of the sample holder that is not pumped down. Furthermore, the film permits an electron beam to pass therethrough (see patent document 1).
- When a sample is inspected, a culture medium is first put into a sample capsule together with cells. The cells are cultivated on the film. Then, the sample capsule is placed into an SEM sample chamber that is in a reduced-pressure ambient. An electron beam is directed at the sample placed within the sample capsule from outside the capsule via the film on the capsule. Backscattered electrons are produced from the irradiated sample. The backscattered electrons pass through the film on the capsule and are detected by a backscattered electron detector mounted in the SEM sample chamber. Consequently, an SEM image is derived.
- However, with this technique, the sample is sealed in the closed space and so it has been impossible to give a stimulus to cells or to manipulate them using a manipulator or pipette. The amount of the culture medium put into the sample capsule is about 15 µℓ. Therefore, as the culture medium evaporates, the salinity concentration rises, making it difficult to culture cells. Where the cells should be observed or inspected in vivo, there arises a problem.
- This problem can be solved by increasing the size of the sample capsule to increase the capacity. However, if the film is damaged either by a stimulation induced by an electron beam or by a mechanical stimulus, a new problem is created. That is, the inside of the apparatus is contaminated with a large amount of culture medium.
- An example of a method of obtaining an SEM image by preparing a film withstanding the pressure difference between vacuum and atmospheric pressure, irradiating a sample with an electron beam via the film, and detecting backscattered electrons produced from the sample in this way is described also in non-patent document 1 (especially, Chapter 1: Introduction).
- Examples in which two films of the structure described above are placed opposite to each other with a sample interposed between the films and in which an image is acquired by a transmission electron microscope are described in
patent documents 2 and 3. Especially,patent document 2 also states a case in which an SEM image of the sample interposed between such films is acquired. - Patent document 4 discloses a sample inspection apparatus equipped with an open-close valve for partitioning the space between a film and primary beam irradiation means within a vacuum chamber in order to permit the sample held on the film to be exchanged quickly and to prevent contamination of the inside of the vacuum chamber.
- [Patent document 1]
JP-T-2004-515049 - [Patent document 2]
JP-A-47-24961 - [Patent document 3]
JP-A-6-318445 - [Patent document 4]
JP-A-2007-292702 - [Non-patent document 1] "Atmospheric scanning electron microscopy", Green, Evan Drake Harriman, Ph.D., Stanford University, 1993
- The resolution of an optical microscope is not high enough to observe very tiny regions of biological cells. Imaging using SEM is required. In order to observe cells by SEM while maintaining the liquid, a sample (cells) cultured on a laboratory dish is sealed into a sample capsule. The sample is irradiated with an electron beam via the film formed on the sample capsule. Thus, the sample is imaged.
- However, the sample capsule is a narrow closed space. Therefore, there is the problem that it has been impossible to directly observe the state of the sample immediately after a stimulus is given from the outside to the sample using a manipulator or pipette. Furthermore, the capacity inside the sample capsule is small. Consequently, when moisture evaporates and the salinity concentration rises, it is difficult to culture cells for a long time inside the sample capsule. Hence, there are problems in observing cells for a long time.
- In an attempt to solve this problem, the present invention is intended to provide an apparatus and method for inspecting a sample in such a way that biological cells held in a liquid state can be manipulated from the outside with a manipulator, pipette, or the like and that consideration is given to long-term observation.
- Patent document 4 states that when a sample is exchanged, the space between the film and the primary beam irradiation means is partitioned off by the open-close valve and that under this condition, only the space on the film side is returned to the normal pressure. It also states that if the film is damaged during inspection of the sample, the valve is closed, partitioning off the space inside the vacuum chamber to thereby prevent contamination into the vacuum chamber.
- With the open-close valve described in patent document 4, however, the space inside the vacuum chamber is partitioned off hermetically. Therefore, it takes a considerable time to open and close the open-close valve. The portion that should be certainly prevented from being contaminated is the inside of the primary beam irradiation means (electron optical column). If it takes a considerable time to open and close the valve, it is not assured that contamination of the inside of the primary beam irradiation means is prevented.
- The present invention relates to a sample inspection apparatus and method free of these problems. It would be desirable to provide a sample inspection apparatus that can be easily maintained and serviced by certainly preventing at least the inside of primary beam irradiation means from being contaminated. It would be further desirable to provide a sample inspection-method implemented by the sample inspection apparatus.
- A sample inspection apparatus according to the present invention has a film including a first surface to hold a sample thereon, a vacuum chamber for reducing the pressure of an ambient in contact with a second surface of the film, primary beam irradiation means which is connected with the vacuum chamber for irradiating the sample with a primary beam via the film, signal detection means for detecting a secondary signal produced from the sample in response to the beam irradiation, a partitioning member , having a receiver dish, located in the vacuum chamber configured to move between two extreme positions, one extreme position in an open state and the other extreme position in a closed state, in the open state the partitioning member does not interrupt an optical axis of the primary beam, in the closed state the partitioning member is moved to a position between the film and the primary beam irradiation means which are located opposite to each other and interrupts an optical axis of the primary beam without hermetically isolating a side on which the film is located from a side on which the primary beam irradiation means is located within the vacuum chamber in order that the receiver dish receives the sample that flows into the vacuum chamber when the film is broken.
- In one aspect of the invention, there is further provided detection means which, if there is any damage to the film, can detect the damage. When the damage to the film is detected, the partitioning member is configured to move to the closed state in a-position between the film and the primary beam irradiation means so that the partitioning member interrupts the optical axis of the primary beam. At the same time, the inside of the primary beam irradiation means and the inside of the vacuum chamber can be returned to the atmospheric pressure. In this case, gas can be supplied into the vacuum chamber via the inside of the primary beam irradiation means.
- The partitioning plate may have a receiver dish structure capable of receiving and stopping the sample flowing into the vacuum chamber when the film is damaged. The first surface may hold the sample in an open state in which access to the surface from the outside is allowed. Furthermore, a manipulator having a front-end portion capable of being brought close to or making contact with the sample and optical image acquisition means for observing the sample and the manipulator may be provided.
- Where there is the detection means which, if there is any damage to the film, can detect the damage, the partitioning plate is automatically operated in response to the detection. The detection means detects a rise in pressure inside the vacuum chamber caused by the damage to the film.
- The first surface of the film may be the upper surface of the film. The second surface of the film may be the lower surface of the film. The primary beam is a beam of charged particles or an electron beam. The secondary signal can be at least one type of secondary electrons, backscattered electrons, X-rays, and cathodoluminescent light.
- An inspection method according to the invention starts with holding a sample on a first surface of a film. The pressure of a space inside the vacuum chamber in contact with a second surface of the film is reduced. The sample is irradiated with a primary beam via the film by primary beam irradiation means. A secondary signal produced from the sample in response to the beam irradiation is detected to inspect the sample. This method is characterized in that when any damage to the film is detected, a partitioning member having a receiver dish interrupts an optical axis of the primary beam by moving to a position between the film and the primary beam irradiation means without hermetically isolating a side on which the film is located from a side on which the primary beam irradiation means is located within the space in order that the receiver dish receives the sample that flows into the vacuum chamber when the film is broken.
- In this method, when any damage to the film is detected, the partitioning plate can partition off the region. Concurrently, the inside of the primary beam irradiation means and the inside of the vacuum chamber can be returned to the atmospheric pressure. In this case, gas can be supplied into the vacuum chamber via the inside of the primary beam irradiation means.
- The partitioning plate may have a receiver dish structure capable of stopping and accepting the sample. The first surfacemay hold the sample in an open state in which access to the surface from the outside is allowed. Furthermore, the sample may be manipulated. In addition, an optical image of the sample may be acquired.
- The damage to the film can be detected based on a rise in pressure in the vacuum chamber caused by the damage to the film. The first surface of the film may be the upper surface of the film. The second surface of the film may be the lower surface of the film. The primary beam is a beam of charged particles or an electron beam. The secondary signal can be at least one type of secondary electrons, backscattered electrons, X-rays, and cathodoluminescent light.
- In the sample inspection apparatus according to the present invention, the partitioning plate partitions off the region across which the film and the primary beam irradiation means are located opposite to each other without hermetically isolating the side on which the film is located from the side on which the primary beam irradiation means is located within the vacuum chamber.
- In the sample inspection method according to the present invention, the partitioning plate partitions off the region across which the film and the primary beam irradiation means are located opposite to each other without hermetically isolating the side on which the film is located from the side on which the primary beam irradiation means is located in the space in contact with the second surface of the film.
- Consequently, if the film is damaged and broken, and if the sample held on the film flows into the vacuum chamber (or, the space), the partitioning plate quickly partitions off the region across which the film and primary beam irradiation means are located opposite to each other. Hence, contamination of at least the primary electron beam irradiation means can be prevented with certainty.
- Accordingly, it is necessary to clean only the inside of the vacuum chamber. The sample inspection apparatus can be maintained and serviced easily. Additionally, damage to the primary electron beam irradiation means, which is expensive, can be prevented.
- Other preferred embodiments of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the invention.
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Fig. 1 is a schematic diagram of a first embodiment of the sample inspection apparatus according to the present invention; -
Fig. 2 is a schematic diagram similar toFig. 1 , but showing a different state; -
Fig. 3 is a cross-sectional view of a sample holder according to the invention; -
Fig. 4 is a schematic perspective view of a framelike member constituting the sample holder according to the invention; -
Fig. 5 shows cross sections illustrating a method of fabricating the framelike member constituting the sample holder according to the invention; -
Fig. 6 is a schematic diagram of a modification of the first embodiment of the sample inspection apparatus according to the invention; -
Fig. 7 is a schematic diagram of a second embodiment of the sample inspection apparatus according to the invention; and -
Fig. 8 is a schematic diagram of a modification of the second embodiment of the sample inspection apparatus according to the invention. - Sample inspection apparatus and method according to the present invention are hereinafter described with reference to the drawings.
-
Fig. 1 is a schematic diagram of a first embodiment of the sample inspection apparatus according to the present invention. The apparatus consists chiefly of anoptical microscope 27, amanipulator 26, and an electronbeam apparatus section 29 located under asample holder 40. The electronbeam apparatus section 29 includes an electronoptical column 1 forming primary beam irradiation means. Anelectron gun 2 forming an electron source is disposed in the electronoptical column 1 and emits an acceleratedelectron beam 7 that is a primary beam. Theelectron beam 7 is one kind of charged particle beam. Thebeam 7 is focused by a condenser lens (objective lens) 3. - The
focused electron beam 7 is directed at aliquid sample 20 via a sample-holding film 32 (described later) formed on thesample holder 40. Thesample 20 is held on thesample holder 40. In the present embodiment, theliquid sample 20 includes biological cells and a culture medium. The upper side of thesample 20 is open and in contact with a normal-pressure (atmospheric-pressure) ambient. - The front-end side of the electron
optical column 1 is connected with avacuum chamber 11. Theelectron gun 2 is mounted in the base side of theoptical column 1. The base side of thecolumn 1 is located under thevacuum chamber 11. Because of this configuration, theelectron beam 7 released from theelectron gun 2 travels upward through theoptical column 1, passes through the space inside thevacuum chamber 11 and through the sample-holdingfilm 32, and reaches theliquid sample 20. - During the irradiation, the
electron beam 7 is deflected by deflection means (not shown). Thus, thebeam 7 scans theliquid sample 20. At this time, a specimen contained in theliquid sample 20 is also scanned with thebeam 7. - The electron
optical column 1 forms the primary beam irradiation means in this way. In the present embodiment, the column is of the inverted type. A backscattered electron detector 4 is mounted on the front-end side of theoptical column 1 inside thevacuum chamber 11. The backscattered electron detector 4 detects backscattered electrons produced when the specimen included in theliquid sample 20 is irradiated with theelectron beam 7. For example, a semiconductor detector using a PN junction or a scintillator detector using a YAG crystal is used as the backscattered electron detector 4. - The output signal from the backscattered electron detector 4 is sent to an
image formation device 22 disposed outside thevacuum chamber 11. Theimage formation device 22 forms image data based on the output signal from the detector 4. The image data corresponds to an SEM image, and is sent to adisplay device 23. Thedisplay device 23 displays an image based on the image data sent in. The displayed image forms an SEM image. If necessary, the image data formed by theimage formation device 22 is sent to acomputer 25. Thecomputer 25 image-processes the image data and makes decisions based on the result of the image processing. - The inside of the electron
optical column 1 is pumped down to a desired pressure by vacuum pumping means 8. The inside of thevacuum chamber 11 is evacuated to a desired pressure by vacuum pumping means 9. Thevacuum chamber 11 is placed over apedestal 10 via a vibration-proofingdevice 13. - A sample
holder placement portion 12 is formed on top of thevacuum chamber 11 and provided with a hole to permit theelectron beam 7 to be directed at the sample-holdingfilm 32. Thesample holder 40 is placed on theplacement portion 12 via an O-ring (not shown). Consequently, thesample holder 40 is withdrawably supported in thevacuum chamber 11. - A
vacuum gauge 15 for detecting the pressure inside thevacuum chamber 11 is mounted in thevacuum chamber 11. A partitioning plate or partitioningmember 14 is mounted between the front end of the electronoptical column 1 and the sample-holdingfilm 32. InFig. 1 , thepartitioning plate 14 is in its open state. If thefilm 32 is damaged and thevacuum gauge 15 has detected a given rise in the pressure, thepartitioning plate 14 automatically moves as shown inFig. 2 . For example, if thefilm 32 is damaged, theliquid sample 20 flows into thevacuum chamber 11, and the pressure inside the vacuum chamber increases above 100 Pa, then thepartitioning plate 14 is moved as shown inFig. 2 . - After the
partitioning plate 14 has been moved, it partitions off the region across which the sample-holdingfilm 32 and the electronoptical column 1 are disposed opposite to each other. Inside the sample chamber, the side on which the sample-holdingfilm 32 is located is not hermetically isolated from the side on which the electronoptical column 1 is located. - Consequently, the
liquid sample 20 entered in thevacuum chamber 11 can be received and stopped by thepartitioning plate 20, thus preventing contamination of the electronoptical column 1 and backscattered electron detector 4. If thepartitioning plate 14 is equipped with a receiver dish that receives liquid sample, then it is possible to cope with inflow of a large amount of liquid sample. - The
partitioning plate 14 vertically partitions the inside of thevacuum chamber 11 into two spaces not completely but partially in this way. Therefore, the structure is simple. The partitioning plate can be operated at high speed. In the present embodiment, the partitioning plate can be set into operation (brought to the state ofFig. 2 from the state ofFig. 1 ) after a lapse of 0.1 second since thevacuum gauge 15 detected a rise in pressure. Furthermore, the partitioning plate can be thinned. Consequently, the distance between the front end of the electronoptical column 1 and the sample-holding film 32 (i.e., the working distance of the SEM) can be reduced, thus achieving high resolution. - The electron
beam apparatus section 29 having the electronoptical column 1,vacuum chamber 11, partitioningplate 14, andvacuum gauge 15 is controlled by anelectron beam controller 24. Themanipulator 26 for giving a stimulus (such as a voltage, chemical substance, or medicine) to the sample and for moving it if necessary and anoptical microscope 27 are placed on the sampleholder placement portion 12. Themicroscope 27 permits one to observe the sample and to check the position of themanipulator 26. These components are controlled by anoverall controller 28. - The optical axis of the
optical microscope 27 is coincident with the optical axis of theelectron beam 7. Alternatively, the center of field of view of theoptical microscope 27 is coincident with the center of field of view of the SEM image. A region observed by the optical microscope can be made substantially coincident with the SEM image. The field of view of the SEM image and the field of view of theoptical microscope 27 can be adjusted by manipulating themanipulator 26 ormoving the sampleholder placement portion 12 on which thesample holder 40 is placed by means of a moving mechanism (not shown). - The inspection apparatus according to the present invention has the electron
beam apparatus section 29,manipulator 26,optical microscope 27,electron beam controller 24,overall controller 28,image formation device 22, anddisplay device 23. These portions are connected with thecomputer 25. Information can be exchanged between these portions. - The
sample holder 40 is constructed as shown inFig. 3 . Thesample holder 40 is composed of a dish-like body portion 37 made of plastic or glass and a film holder (framelike member) 18 on which the sample-holdingfilm 32 is formed. Thefilm 32 transmits theelectron beam 7. A recessed portion is formed inside thebody portion 37. The bottom surface of the recessed portion forms a sample-holdingsurface 37a that is open. - The sample-holding
surface 37a of thebody portion 37 is (centrally in the example ofFig. 3 ) provided with a through-hole 37b. Astep portion 37c is formed around thehole 37b on the side of the sample-holdingsurface 37a. Thefilm holder 18 is disposed on thestep portion 37c and has the sample-holdingfilm 32. The sample-holdingfilm 32 has afirst surface 32a that forms the sample-holdingsurface 37a. The sample-holdingsurface 37a is substantially flush with the sample-holdingsurface 37a of thebody portion 37. Consequently, at least a part of the sample-holdingsurface 37a of thesample holder 40 is formed by the sample-holdingfilm 32. - Tapering
portions 37d are formed on the side of thehole 37b on the opposite side of the sample-holdingsurface 37a. The taperingportions 37d are spread apart toward the surface on the opposite side of the sample-holdingsurface 37a. The spread angle is set to 90° to 120°. - A region of the lower surface of the
sample holder 40 might be exposed to a vacuum ambient and become irradiated with theelectron beam 7. Aconductive film 301 is formed on this region to prevent charging of thesample holder 40 when it is irradiated with thebeam 7. Theconductive film 301 is in contact with thefilm holder 18 . Electric charge accumulated by being irradiated by theelectron beam 7 can be dissipated away to theliquid sample 20 via thefilm holder 18 made of silicon. The presence of theconductive film 301 reduces the charging of the lower surface of thesample holder 40 and can prevent displacement of the orbit of the beam 7 (that would normally be produced when theliquid sample 20 is illuminated with the beam 7) and distortion and illumination spots in the SEM image that would be normally produced by displacement of the orbit of backscattered electrons. - Accumulation of electric charge can be prevented with certainty by connecting a grounding line to the
liquid sample 20 or electrically connecting theconductive film 301 with the sampleholder placement portion 12. Theconductive film 301 can be formed, for example, by vapor-depositing aluminum or gold or applying silver paste. - The structure of the
film holder 18 is shown inFig. 4 . The sample-holdingfilm 32 is formed on asilicon substrate 34. Thefirst surface 32a of the sample-holding film 32 (lower surface as viewed inFig. 4 ; upper surface as viewed inFig. 3 ) is exposed. Theliquid sample 20 containing liquid such as a culture medium and sample cells is placed on the first surface (sample-holding surface) 32a of the sample-holdingfilm 32. Since thefirst surface 32a is under atmospheric pressure, evaporation of moisture from theliquid sample 20 can be suppressed to a minimum. - The
silicon substrate 34 is centrally provided with anopening 34a (upper surface inFig. 4 ; lower surface inFigs. 1 and3 ) covered with the sample-holdingfilm 32. A central portion of the second surface 32b of the sample-holdingfilm 32 is exposed to the inside ambient of thevacuum chamber 11 through theopening 34a. Thefirst surface 32a of the sample-holdingfilm 32 is exposed to the atmospheric-pressure ambient, while the second surface 32b is exposed to the vacuum ambient. In order to withstand the pressure difference, thefilm 32 is supported and reinforced with alattice 35. - A method of creating the
film holder 18 is next described by referring toFig. 5 . First, as shownFig. 5(a) , silicon nitride,films silicon substrate 501 using CVD (chemical vapor deposition). A typical thickness of thefilms silicon nitride films Fig. 5(b) ). The layer of resist 505 is patterned photolithographically to leave behind resistlayer portions 505a (Fig. 5(c) ). Using the resist pattern as a mask, thesilicon nitride film 503 is processed by dry etching, and siliconnitride film portions 503a are left behind (Fig. 5(d) ). - Using the pattern as a mask, the
silicon substrate 501 is wet-etched with KOH to form an opening 510 (Fig. 5(e) ). The resistlayer portions Fig. 5(f) ). Where thelattice 35 is not present, thefilm holder 18 is completed at this point. Resist 506 is applied on the layer of silicon nitride film 502 (Fig. 5(g) ). A layer ofmetal 507 of A1 or Ni is formed to a thickness of 1 µm on the opposite side of the silicon nitride film 502 (Fig. 5(h) ). Resist 508 is applied on themetal layer 507, and a pattern is photolithographically formed using a mask (Fig. 5 (i) ). Using the resistlayer 508 as a mask, themetal layer 507 is etched (Fig. 5(j) ). Finally, the resistlayer 508 is removed by ashing or organic cleaning (Fig. 5(k) ). As a result, theopening 34a andlattice 35 are formed. - The
film holder 18 fabricated in this way is inverted up and down from the state ofFig. 4 . Thefirst surface 32a of thesilicon nitride film 502 that is the sample-holdingfilm 32 is taken as an upper surface. The second surface 32b can also be taken as an upper surface. - The
film holder 18 is firmly attached to thestep portion 37c over thehole 37b formed in thebody portion 37 forming thesample holder 40. Thus, thesample holder 40 is fabricated (Fig. 3 ). To attach theholder 18 to the step portion firmly, bonding using an epoxy-based or silicone-based adhesive or fusion making use of heat, ultrasonic waves, or laser light can be used. Consequently, thefilm holder 18 is firmly held in a position corresponding to thehole 37b in the sample-holdingsurface 37a of thebody portion 37. - In the present embodiment, the
body portion 37 andfilm holder 18 are combined to fabricate thesample holder 40. The sample-holdingfilm 32 may be directly firmly bonded to thebody portion 37. Thebody portion 37 and the sample-holdingfilm 32 may be fabricated integrally. Furthermore, cell adhesion molecules (described later) acting as molecules for bonding the sample may be applied to the sample-holdingsurface 37a including thefirst surface 32a of the sample-holdingfilm 32. - The thickness of the
silicon nitride film 502 is set to a range of from 10 to 1,000 nm. The sample-holdingfilm 32 of thefilm holder 18 is made of silicon nitride. In addition, thefilm 32 may bemade of silicon oxide, boron nitride, polymer, polyethylene, polyimide, polypropylene, orcarbon. Where films of these materials are used, their film thicknesses are set to a range of from 10 to 1,000 nm. The sample-holdingfilm 32 made of the aforementioned material transmits theelectron beam 7 but does not transmit gas or liquid. Moreover, it is necessary that the film be capable of withstanding a pressure difference of at least 1 atmosphere across the opposite surfaces. - As the thickness of the sample-holding
film 32 is reduced, scattering of theelectron beam 7 is reduced and, therefore, the resolution is improved but the film is more easily damaged. As the thickness is increased, scattering of theelectron beam 7 increases, resulting in decreased resolution. However, the film is less likely to be damaged. The preferable thickness of the film is 20 to 200 nm. - A sample inspection method according to the present invention is next described. First, as shown in
Fig. 3 ,cells 38 forming a sample are cultured within aculture medium 39 using thesample holder 40. In order to culture thecells 38 in this way, it is necessary to graft the sample cells from the laboratory dish where they have been previously cultured to thesample holder 40. For this purpose, a normal method as described below is used. - The culture medium is Product No. D5796 of Sigma-Aldrich Co., for example. First, the culture medium is discarded from the laboratory dish where the cells have been previously cultured. A mixture liquid of trypsin and EDTA (ethylenediaminetetraacetic acid) is put into the dish to peel off the cells adsorbed to the dish. The peeled cells are recovered into a centrifuge tube. A culture medium is put into the tube. The trypsin is inactivated and then the cells are spun down. Then, the supernatant fluid is discarded from the centrifuge tube and the remaining liquid is stirred in the culture medium. Apart (e.g., 1/10) of the stirred liquid including the
cells 38 is entered into thesample holder 40. The culture medium (liquid sample) 39 is grafted. - Under this condition, the holder is allowed to stand still in a cell culture chamber. After a lapse of several hours, the
cells 38 begin to be adsorbed onto the sample-holdingsurface 37a of thesample holder 40 including thesurface 32a of the sample-holdingfilm 32 and proliferate. The aforementioned method may be modified according to cells, and is merely one example. Consequently, thecells 38 which are to be observed or inspected and become a sample are cultured within thesample holder 40. It follows that theliquid sample 20 containing thecultured cells 38 andculture medium 39 is constituted. - Depending on biological cells, if cell adhesion molecules (molecules for bonding of the sample) are applied to the sample-holding
surface 37a of the sample holder 40 (especially, the first surface (sample-holding surface) 32a of the sample-holdingfilm 32 observed with an electron beam), cultivation is facilitated. The cell adhesion molecules cause cells arranged for cultivation and cells proliferated by cultivation to be adsorbed onto the sample-holding surface. Examples of the cell adhesion molecules include collagen, fibronectin, vitronetin, cadherin, integrin, claudins, desmogleins, neuroligin, neurexin, selectin, laminins, and poly-L-lysine. - After the cells are cultured within the
sample holder 40 as described above, thesample holder 40 is placed on theholder placement portion 12. At this time, thepartitioning plate 14 is closed and in the state ofFig. 2 . Subsequently, the insides of thevacuum chamber 11 and electronoptical column 1 are evacuated to desired degrees of vacuum using the vacuum pumping means 8 and 9. For example, the pressure inside of the electronoptical column 1 is set below 1 Pa. The pressure inside the electron optical column 1 (especially, around the electron gun 2) is set to about 10-4 to 10-5 Pa, for example. - The positions of the
cells 38 and of themanipulator 26 are then checked with theoptical microscope 27. A glass microtube holding microelectrodes therein is installed at the front end of the manipulator. A voltage can be applied to the cells through the microelectrodes. A liquid can be made to flow in and out through the glass microtube for manipulation. - Under this condition, the
manipulator 26 is moved while making an observation with theoptical microscope 27 to bring thecells 38 close to the glass microtube. Then, a negative pressure is applied to the glass microtube to bring it into intimate contact with the cell membranes. As a result, potential response can be measured. - When the
manipulator 26 is moved as described above, if the sample-holdingfilm 32 is erroneously damaged or destroyed, and if theliquid sample 20 flows into thevacuum chamber 11, thesample 20 can be received and stopped by thepartitioning plate 14 because the plate is closed. Consequently, the electronoptical column 1 is not contaminated. Where thepartitioning plate 14 is not present as in the prior art, theliquid sample 20 enters the electronoptical column 1, making it necessary to clean or replace the column. As a result, the apparatus is made usable. - We now return to the observation sequence. After checking that the sample-holding
film 32 on which theliquid sample 20 is placed has not been damaged, thepartitioning plate 14 is opened. Thus, the inside of thevacuum chamber 11 is ceased to be partitioned. Thereafter, in order to prevent light from entering the backscattered electron detector 4 via the sample-holdingfilm 32, the light illumination of theoptical microscope 27 is ceased. Other extraneous light is blocked in a manner not shown. The blocking also shields thefilmholder 18 andliquid sample 20 against radiation rays produced when theelectron beam 7 hits thefilm holder 18 andsample 20. - Then, as shown in
Fig. 1 , theelectron beam 7 is directed at theliquid sample 20 including thecells 38 from the electronoptical column 1 to perform imaging. Thebeam 7 passes through the sample-holdingfilm 32 of thesample holder 40 and hits thecells 38. Backscattered electrons produced from thecells 38 in response to the illumination are detected by the backscattered electron detector 4. - Since the
aforementioned tapering portions 37d are formed around thehole 37b of thebody portion 37 forming thesample holder 40, collision of the backscattered electrons against the inner side surface of thehole 37b can be suppressed to a minimum. That is, the backscattered electrons can be suppressed from being blocked. The backscattered electrons can be detected efficiently by the backscattered electron detector 4. - A detection signal produced from the backscattered electron detector 4 is fed to the
image formation device 22, which in turn forms image data based on the detection signal. Based on the image data, an image (SEM image) is displayed on thedisplay device 23. - Subsequently, an electrical stimulus is given to the
cells 38 using the microelectrodes installed at the front end of themanipulator 26 to manipulate the cells. An SEM image is acquired in the same way as in the above-described process step. The response of thecells 38 to the stimulus is checked. - After the imaging, the
partitioning plate 14 is closed to prevent contamination of the electronoptical column 1 if the sample-holdingfilm 32 should be damaged. Before a variation caused by application of a stimulus to thecells 38 is observed by SEM as described above, an observation may be made with theoptical microscope 27. Also, at this time, if thepartitioning plate 14 is closed, risk of contamination occurring when the sample-holdingfilm 32 is broken can be reduced. - In any case, if the
partitioning plate 14 is closed when theelectron beam 7 is not directed at theliquid sample 20, the probability of contamination of the inside of the apparatus can be reduced by shortening the interval for which thepartitioning plate 14 is opened during inspection. - Where the speed of reaction of the
cells 38 to the stimulus is low, thepartitioning plate 14 may be once closed. Theplate 14 may be again opened at a time when a reaction is deemed to have taken place. Then, imaging may be performed using theelectron beam 7. The reaction can be checked with theoptical microscope 27. - The
manipulator 26 can have a mechanism capable of spraying a chemical substance or medicine into theliquid sample 20. Behavior of thecells 38 in response to the chemical substance or medicine can be observed or inspected while observing the cells by SEM. Furthermore, a function of permitting a liquid to flow out can be imparted to themanipulator 26. This permits the sprayed substance to be recovered. Also, the pH of the culture medium and the osmotic pressure can be maintained constant. - In the foregoing, backscattered electrons are used to form an image. Backscattered electrons produce a signal intensity proportional to the atomic number. Therefore, where the sample is almost totally made of substances of low atomic numbers such as a biological sample, the image contrast is very low, and it is difficult to improve the resolution.
- Accordingly, a heavy metal such as gold may be adsorbed onto portions of the
cells 38 to be noticed in their behavior. In particular, gold is adsorbed onto the portions (antigen) via an antibody by causing the antigen tagged with gold particles having the nature of being adsorbed on the portions (antigen) to be sprayed against the cells by making use of an antigen-antibody reaction. Furthermore, a fluorescent dye or quantum dots (e.g., nanoparticles of Si or particles of CdSe coated with ZnS and having sizes of 10 to 20 nm) that emit light when irradiated with an electron beam may be previously adsorbed onto certain portions of thecells 38, and the emitted light may be observed with an optical microscope. - In the above embodiment, normally used gold particles have particle diameters of 10 to 30 nm. However, the adsorptive force between the antibody and gold particles is weak, and gold particles of 10 to 30 nm may not be attached. In this case, very small gold particles (nanogold particles) having particle diameters of the order of nanometers are first attached to the antibody. Under this condition, the gold particles are too small and it is difficult to observe them by SEM. Silver is adsorbed around the gold particles by making use of a silver sensitizer. This makes it easier to detect them by SEM.
- In the foregoing, cells previously cultured in a laboratory dish are taken out and grafted onto the
sample holder 40. Then, the cells are cultured. Alternatively, cells may be taken from a living organism and directly placed on the sample-holdingsurface 37a of thesample holder 40. The cells may be cultured in thesample holder 40. - As described so far, the present invention makes it possible to observe a specimen by SEM via the sample-holding
film 32, the specimen being included in a liquid. Especially, the use of an open sample chamber facilitates giving a stimulus to (or, manipulating) cells using the manipulator because access to the sample can be made from the outside. - During observation with the SEM under the condition where the
partitioning plate 14 is opened as shown inFig. 1 , if the sample-holdingfilm 32 is broken, theliquid sample 20 flows into thevacuum chamber 11, increasing the pressure. If thevacuum gauge 15 has detected the increased pressure to be higher than 100 Pa, for example, information about it is sent to theelectron beam controller 24, and an instruction for closing thepartitioning plate 14 is sent to thepartitioning plate 14. - As a result, the
partitioning plate 14 is moved as shown inFig. 2 . Theliquid sample 20 is received and stopped by thepartitioning plate 14. Where the amount of theliquid sample 20 is large, it is possible to prepare a receiver dish on thepartitioning plate 14. It takes only 0.1 second until thepartitioning plate 14 is closed after detection of a pressure rise. The contamination of the electronoptical column 1 can be reduced to a level at which no cleaning is necessary. In this way, the present invention enhances convenience in use of the apparatus. Furthermore, the amount of culture medium can be increased. Cells are allowed to survive for a long time. SEM imaging can be performed for a long time. - In the present invention, the inverted type SEM is used. Depending on samples, a normal non-inverted type SEM using a sealed sample capsule as described in the "Description of the Related Art" of this specification can be used without problem. In this case, also, the
partitioning plate 14 needs to be located between the sealed sample capsule and the front end of the electronoptical column 1. - In the above embodiments, an electron beam is used as the primary beam. If the sample-holding
film 32 shows sufficient shock resistance and strength against impingement of other charged particle beam such as a helium ion beam, the invention can also be applied in a case where the other charged particle beam is used. - In the above embodiments, backscattered electrons are used as a secondary signal. Information about the
cells 38 can also be obtained by detecting other form of information such as secondary electrons, X-rays, cathodoluminescent light, and electric current absorbed into thecells 38 forming a sample. It is convenient to use themanipulator 26 in measuring the absorption current. - It is required that the sample-holding
film 32 of the present embodiment withstand a pressure difference of at least 1 atm. and that gas or liquid do not flow in or out. Specifically, the material of thefilm 32 includes at least one of polymer, polyethylene, polyimide, polypropylene, carbon, silicon oxide, silicon nitride, and boron nitride. - In the above embodiments, the
partitioning plate 14 and backscattered electron detector 4 are fabricated separately. As a modified embodiment, they may be integrated as shown inFig. 6 . That is, in the embodiment ofFig. 6 , the backscattered electron detector 4 is mounted at the front end of thepartitioning plate 14. In this structure, when thepartitioning plate 14 is opened, the backscattered electron detector 4 is located immediately above the electronoptical column 1, maximizing the efficiency at which backscattered electrons are detected. - A second embodiment of the sample inspection apparatus of the present invention is next described by referring to
Fig. 7 . The sample inspection apparatus shown inFig. 7 inspects a sample by irradiatingcells 110 held on a sample-holdingfilm 150 with an electron beam from below via thefilm 150. - The sample-holding
film 150 is held in alaboratory dish 130. Asample 140 consisting of a liquid culture medium and thecells 110 is held in thedish 130. Under this condition, thedish 130 is placed over avacuum chamber 230 via an O-ring 160. An opening is formed in an upper part of thevacuum chamber 230 and located opposite to the sample-holdingfilm 150 in thelaboratory dish 130. - The front end of an electron
optical column 200 acting as primary beam irradiation means is connected with a lower part of thevacuum chamber 230. Thecolumn 200 is controlled by anSEM control circuit 205. Correspondingly, an electron beam is emitted from anelectron gun 2500 disposed in the electronoptical column 200 toward thesample 140. - When the electron beam is made to impinge on the
cells 110 via the sample-holdingfilm 150, backscattered electrons are produced from thecells 110 and detected by a backscatteredelectron detector 210. The output signal from thedetector 210 based on the detected backscattered electrons is amplified by apreamplifier 215 and accepted into acontrol computer 1300 via theSEM control circuit 205. - A two-dimensional image based on a signal from the
control computer 1300 is displayed as a sample image on adisplay monitor 1400. AnX-ray shielding cover 250 is mounted to prevent X-rays produced from the vicinities of the sample in response to the electron beam irradiation from leaking from the apparatus. The ambients inside and outside thecover 250 are at normal pressure (atmospheric pressure). - The inside of the
vacuum chamber 230 is evacuated via atube 231 by avacuum pump 2100. Thetube 231 is connected with the bottom surface of the inside of thevacuum chamber 230 as shown. Thepump 2100 is connected with thetube 231 via avalve 2105. - A
pressure gauge 2010 acting as pressure detection means is connected with thetube 231. A leakinggas supply source 2220 is connected also with thetube 231 via anothervalve 2210. Thepressure gauge 2010 can detect the pressure inside thevacuum chamber 230 via thetube 231. - The
pressure gauge 2010,vacuum pump 2100, andvalves vacuum control system 2000. - On the other hand, the inside of the electron
optical column 200 is evacuated by avacuum pump 2504 via atube 232. Thetube 232 is connected into thecolumn 200 as shown. Thepump 2504 is connected with thetube 232 via avalve 2503. A leakinggas supply source 2502 is connected with thetube 232 via avalve 2501. Thevacuum pump 2504 andvalves vacuum control system 2000. - The backscattered
electron detector 210 is mounted at the front end of the partitioning plate or partitioningmember 260. Thepartitioning plate 260 is driven by a partitioning plate drive means 265, which in turn is controlled in operation by thevacuum control system 2000. Thedetector 210 is provided with an opening to permit passage of the electron beam coming from the electronoptical column 200. - In the sample inspection apparatus constructed as described so far, when the
cells 110 are observed or inspected, the insides of thevacuum chamber 230 and electronoptical column 200 are pumped down to set degrees of vacuum. - Specifically, the
vacuum control system 2000 closes thevalve 2210 and opens thevalve 2105. The inside of thevacuum chamber 230 is evacuated by thevacuum pump 2100 via thetube 231. - The
vacuum control system 2000 closes thevalve 2501 and opens thevalve 2503. Consequently, the inside of the electronoptical column 200 is evacuated by thevacuum pump 2504 via thetube 232. - Under this condition, an electron beam is directed at the
cells 110 forming a sample from theelectron gun 2500 in the electronoptical column 200 through thevacuum chamber 230. The beam reaches thecells 110 through the sample-holdingfilm 150. Backscattered electrons produced from thecells 110 at this time pass through the sample-holdingfilm 150 and reach the backscatteredelectron detector 210, where the electrons are detected. As a result, a sample image is formed. - During the observation or inspection of the sample as described above, if the sample-holding
film 150 holding thesample 140 including the liquid andcells 110 is damaged or destroyed, atmosphere enters through the damaged portion of the film. The degree of vacuum of the ambient inside thevacuum chamber 230 deteriorates. For example, when the inside pressure is set to 1 Pa, the pressure increases rapidly to above 30 Pa. In this case, the vacuum deterioration is detected by thepressure gauge 2010, and information about it is sent to thevacuum control system 2000. - When the information is detected, the
vacuum control system 2000 operates the partitioning plate drive means 2 65. Consequently, thepartitioning plate 260 moves through thevacuum chamber 230 and is placed between the sample-holdingfilm 150 and the electronoptical column 200. At this time, thepartitioning plate 260 partitions off the region across which the sample-holdingfilm 150 and electronoptical column 200 are located opposite to each other without hermetically isolating the side where the sample-holdingfilm 150 is located from the side where thecolumn 200 is located inside thevacuum chamber 230. - Consequently, the
partitioning plate 260 can be instantly moved at high speed. If the sample-holdingfilm 150 is damaged and thesample 140 including liquid intrudes into thevacuum chamber 230, most of the entering sample is received and stopped by thepartitioning plate 260. - Simultaneously with the above-described operation, the
vacuum control system 2000 closes thevalve 2105 on the side of thetube 231 and thevalve 2503 on the side of thetube 232 and opens thevalve 2501 on the side of thetube 232. Consequently, vacuum pumping through thetube 231 is stopped. Also, vacuum pumping through thetube 232 is stopped. Leaking gas such as nitrogen from thegas supply source 2502 is supplied into the electronoptical column 200 via thetube 232. - The leaking gas supplied into the electron
optical column 200 reaches the inside of thevacuum chamber 230. The inside of thecolumn 200 and the inside of thevacuum chamber 230 including the inside of thetube 231 are returned to the atmospheric pressure. At this time, the leaking gas flows into thevacuum chamber 230 from inside thecolumn 200 via the front end of thecolumn 200 and so if the sample-holdingfilm 150 is damaged and thesample 140 containing liquid enters thevacuum chamber 230, thesample 140 does not enter thecolumn 200. Consequently, contamination is prevented. - Where it is desired to return the inside of the
vacuum chamber 230 to the atmospheric pressure quickly, the leaking gas can also be supplied from thegas supply source 2220 into thevacuum chamber 230 via thetube 231 by opening thevalve 2210 on the side of thetube 231. In this case, it is desired to set the flow rate of the leaking gas supplied from thegas supply source 2220 smaller as compared with the flow rate of the leaking gas supplied from thegas supply source 2502 into thecolumn 200 because the flow of the leaking gas from inside thecolumn 200 into thevacuum chamber 230 should be maintained. - In order to prevent the pressure inside the
vacuum chamber 230 from becoming higher than the atmospheric pressure by the supply of the leaking gas (i.e., back pressure is created), thevacuum control system 2000 may close thevalves pressure gauge 2010 has detected that the pressure inside thevacuum chamber 230 has reached a pressure slightly lower than the atmospheric pressure. - In this way, a sample inspection apparatus according to the present invention has a sample-holding film (32, 150) including a first surface on which a sample is held, a vacuum chamber (11, 230) for reducing the pressure of an ambient in contact with a second surface of the film, primary beam irradiation means (1, 200) connected with the vacuum chamber and irradiating the sample with a primary beam via the film, signal detection means (4, 210) for detecting a secondary signal produced from the sample in response to the beam irradiation, a partitioning member (14, 260) capable of partitioning off a region across which the film and the primary beam irradiation means (1, 200) are located opposite to each other without hermetically isolating the side on which the film is located from the side on which the primary beam irradiation means (1, 200) is located inside the vacuum chamber.
- The detection means (15, 2010) is provided which, if the film (32, 150) is damaged, detects the damage. When the detection means has detected the damaged to the film, the partitioning member (14, 260) can partition off the region.
- Furthermore, there is provided the pressure restoration means (2220, 2502) which, when the detection means (15, 2010) has detected any damage to the film, returns the inside of the primary beam irradiation means (1, 200) and the inside of the vacuum chamber (11, 230) to the atmospheric pressure.
- In this structure, when the detection means (15, 2010) has detected any damage to the film, the pressure restoration means (2502) supplies leaking gas into the vacuum chamber (230) via the inside of the primary beam irradiation means (200), whereby the insides of the primary beam irradiation means (200) and vacuum chamber (230) can be returned to the atmospheric pressure. The detection means (15, 2010) can detect any damage to the film based on a rise in pressure inside the vacuum chamber (11, 230).
- The partitioning member (14, 260) can have a receiver dish structure.
- The signal detection means (4, 210) can be mounted to the partitioning member (14, 260).
- The first surface of the sample-holding surface can hold a sample under an open state to permit a manipulator to make access to the surface from the outside. The apparatus can have the manipulator whose front end can be brought close to or make contact with the sample held on the first surface of the film and optical image acquisitionmeans for observing the sample and the manipulator.
- The first surface of the sample-holding film can be taken as the upper surface of the film, while the second surface of the film can be taken as the lower surface of the film. The primary beam released from the primary beam irradiation means (1, 200) can be a beam of charged particles or electron beam. The secondary signal can be at least one type of secondary electrons, backscattered electrons, X-rays, and cathodoluminescent light.
- The sample inspection method according to the present invention can be implemented by inspecting a sample with the above-described sample inspection apparatus.
- The sample inspection method according to the invention starts with holding a sample on a first surface of a sample-holding film (32, 150). The pressure of a space in contact with a second surface of the film is reduced. The sample is irradiated with a primary beam via the film by primary beam irradiation means (1, 200). A secondary signal produced from the sample in response to the primary beam irradiation is detected, thus inspecting the sample. When any damage to the film is detected, the region across which the film and the primary beam irradiation means (1, 200) are located opposite to each other can be partitioned off by the partitioning member (14, 260) without hermetically isolating the side on which, the film is located from the side on which the primary beam irradiation means (1, 200) is located inside the space.
- When any damage to the film is detected, the inside of the primary beam irradiation means (1, 200) and the space can be returned to the atmospheric pressure. When any damage to the film is detected, the inside of the primary beam irradiation means (1, 200) and the space can be returned to the atmospheric pressure by supplying leaking gas into the space via the inside of the primary beam irradiation means (1, 200). The damage to the film can be detected based on a rise in pressure inside the space.
- Additionally, the sample held on the first surface of the film can be manipulated. An optical image of the sample can be acquired.
- Finally, a modification of the second embodiment is shown in
Fig. 8 . The differences between the sample inspection apparatus shown inFig. 8 and the apparatus shown inFig. 7 are that the lower surface of thepartitioning plate 260 is located slightly below the lower surface of the partitioning plate shown inFig. 7 and that an 0-ring 161 is mounted to the lower surface. - In this modification, when the partitioning plate-driving means 265 operates, the
partitioning plate 260 closes the front end of the electronoptical column 200 via the O-ring 161. Consequently, the inside of thecolumn 200 is hermetically isolated from the inside of thevacuum chamber 230. - In this structure, after the
partitioning plate 260 is closed as described above, leaking gas is supplied from the bottom side of thevacuum chamber 230 via thetube 231. In this structure, too, contamination into the electronoptical column 200 can be prevented.
Claims (19)
- A sample inspection apparatus comprising:a film (32) having a first surface (32a) to hold a sample (20) thereon;a vacuum chamber (11) for reducing pressure of an ambient in contact with a second surface (32b) of the film;primary beam irradiation means (1, 200), which is connected with the vacuum chamber, for irradiating the sample with a primary beam via the film; andsignal detection means (4) for detecting a secondary signal produced from the sample in response to the primary beam irradiation;characterised in that the apparatus further comprises:a partitioning member (14), having a receiver dish, located in the vacuum chamber configured to move between two extreme positions, one extreme position in an open state and the other extreme position in a closed state, wherein in the open state the partitioning member does not interrupt an optical axis of the primary beam, and in the closed state the partitioning member is arranged in a position between the film and the primary beam irradiation means which are located opposite to each other to interrupt the optical axis of the primary beam without hermetically isolating a side on which the film is located from a side on which the primary beam irradiation means is located inside the vacuum chamber in order that the receiver dish receives the sample that flows into the vacuum chamber when the film is broken.
- A sample inspection apparatus as set forth in claim 1, wherein there is further provided detection means (15, 2010) for detecting any damage to the film, and wherein the detection means is configured so that when the detection means has detected the damage to the film, the partitioning member is configured to move to the closed state in a position between the film and the primary beam irradiation means so that the partitioning member interrupts the optical axis of the primary beam.
- A sample inspection apparatus as set forth in claim 2, wherein there is further provided pressure restoration means (2220, 2502) which, when said detection means has detected the damage to the film, is configured to return the inside of the primary beam irradiation means and the inside of the vacuum chamber to atmospheric pressure.
- A sample inspection apparatus as set forth in claim 3, wherein when said detection means has detected the damage to the film, said pressure restoration means is configured to return the inside of the primary beam irradiation means and the inside of the vacuum chamber to atmospheric pressure by supplying gas into the vacuum chamber via the primary beam irradiation means.
- A sample inspection apparatus as set forth in any one of claims 2 to 4, wherein said detection means is configured to detect the damage to the film based on a rise in pressure inside the vacuum chamber.
- A sample inspection apparatus as set forth in any one of claims 1 to 5, wherein said signal detection means is mounted on said partitioning member.
- A sample inspection apparatus as set forth in any one of claims 1 to 6, wherein said first surface of the film holds the sample in an open state to permit access to the surface from the outside.
- A sample inspection apparatus as set forth in any one of claims 1 to 7, further comprising:a manipulator (26) having a front-end portion capable of being brought close to or making contact with the sample held on the first surface of the film; andoptical image acquisition means (27) for observing the sample and the manipulator.
- A sample inspection apparatus as set forth in any one of claims 1 to 8, wherein the first surface of said film is an upper surface of the film, while the second surface of the film is a lower surface of the film.
- A sample inspection apparatus as set forth in any one of claims 1 to 9, wherein said primary beam is a beam of charged particles or an electron beam, and wherein said secondary signal is at least one type of secondary electrons, backscattered electrons, X-rays, and cathodoluminescent light.
- A sample inspection method for inspecting a sample using- a sample inspection apparatus as set forth in any one of claims 1 to 10.
- A sample inspection method comprising the steps of:holding a sample (20) on a first surface (32a) of a film (32);reducing pressure of a space in contact with a second surface (32b) of the film, the space being located in an inside of a vacuum chamber (11);irradiating the sample with a primary beam by primary beam irradiation means (1, 200) via the film, the primary irradiation means and the film being located opposite to each other; andinspecting the sample by detecting a secondary signal produced from the sample in response to the primarybeam irradiation;characterized in that the method further comprises:causing a partitioning member (14) having a receiver dish to interrupt an optical axis of the primary beam by moving to a position between the film and the primary beam irradiation means without hermetically isolating a side on which the film is located from a side on which the primary beam irradiation means is located inside the space when any damage to the film is detected in order that the receiver dish receives the sample that flows into the vacuum chamber when the film is broken.
- A sample inspection method as set forth in claim 12, wherein when any damage to said film is detected, inside of said primary beam irradiation means and said space are returned to atmospheric pressure.
- A sample inspection method as set forth in claim 13, wherein when any damage to said film is detected, the inside of said primary beam irradiation means and said space are returned to the atmospheric pressure by supplying gas into said space via the inside of the primary beam irradiation means.
- A sample inspection method as set forth in any one of claims 12 to 14, wherein the damage to said film is detected based on a rise in pressure inside said space.
- A sample inspection method as set forth in any one of claims 12 to 15, wherein said first surface of the film holds the sample in an open state to permit access to the film from the outside.
- A sample inspection method as set forth in any one of claims 12 to 16, wherein the sample held on the first surface of said film is manipulated, and wherein an optical image of the sample is acquired.
- A sample inspection method as set forth in any one of claims 12 to 17, wherein the first surface of said film is an upper surface of the film, while the second surface of the film is a lower surface of the film.
- A sample inspection method as set forth in any one of claims 12 to 18, wherein said primary beam is a beam of charged particles or an electron beam, and wherein said secondary signal is at least one type of secondary electrons, backscattered electrons, X-rays, and cathodoluminescent light.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2008080186A JP5215701B2 (en) | 2008-03-26 | 2008-03-26 | Sample inspection apparatus and sample inspection method |
Publications (2)
Publication Number | Publication Date |
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EP2105727A1 EP2105727A1 (en) | 2009-09-30 |
EP2105727B1 true EP2105727B1 (en) | 2016-03-16 |
Family
ID=40888240
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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EP09250792.0A Not-in-force EP2105727B1 (en) | 2008-03-26 | 2009-03-20 | Scanning electron microscope comprising a film for holding a sample and a dish for receiving sample material from a damaged film |
Country Status (3)
Country | Link |
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US (1) | US8119994B2 (en) |
EP (1) | EP2105727B1 (en) |
JP (1) | JP5215701B2 (en) |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1936363A3 (en) * | 2006-12-19 | 2010-12-08 | JEOL Ltd. | Sample inspection apparatus, sample inspection method, and sample inspection system |
JP5318364B2 (en) * | 2007-01-31 | 2013-10-16 | 日本電子株式会社 | SAMPLE HOLDER, SAMPLE INSPECTION DEVICE, SAMPLE INSPECTION METHOD, AND SAMPLE HOLDER MANUFACTURING METHOD |
EP2601477B1 (en) | 2010-08-02 | 2021-09-22 | Oxford Instruments America, Inc. | Method for acquiring simultaneous and overlapping optical and charged particle beam images |
TWI472751B (en) * | 2011-05-03 | 2015-02-11 | Hermes Microvision Inc | Charged particle system for reticle / wafer defects inspection and review |
US9373480B2 (en) | 2013-04-12 | 2016-06-21 | Hitachi High-Technologies Corporation | Charged particle beam device and filter member |
US9842724B2 (en) | 2015-02-03 | 2017-12-12 | Kla-Tencor Corporation | Method and system for imaging of a photomask through a pellicle |
JP2017174504A (en) * | 2016-03-18 | 2017-09-28 | 株式会社日立ハイテクサイエンス | Composite charged particle beam device |
CN106770405A (en) * | 2016-12-09 | 2017-05-31 | 清华大学 | Ultraphotic diffraction imaging device under a kind of complete atmospheric pressure |
US10921268B1 (en) * | 2019-09-09 | 2021-02-16 | Fei Company | Methods and devices for preparing sample for cryogenic electron microscopy |
CN114280092B (en) * | 2020-09-28 | 2023-07-28 | 华南师范大学 | In-situ observation crystal growth method and observation device |
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JPS4724961U (en) | 1971-04-14 | 1972-11-20 | ||
JPS522785A (en) * | 1975-06-24 | 1977-01-10 | Shimadzu Corp | Analyzing method and its device for the same |
JPH01103249U (en) * | 1987-12-28 | 1989-07-12 | ||
JP2781320B2 (en) | 1993-01-18 | 1998-07-30 | 株式会社蛋白工学研究所 | Sample holder for electron microscope |
JPH1064467A (en) * | 1996-08-23 | 1998-03-06 | Toshiba Corp | Electron microscope |
AU2101902A (en) | 2000-12-01 | 2002-06-11 | Yeda Res & Dev | Device and method for the examination of samples in a non-vacuum environment using a scanning electron microscope |
JP4028255B2 (en) * | 2002-02-26 | 2007-12-26 | ソニー株式会社 | Electron beam irradiation apparatus and electron beam irradiation method |
US7230242B2 (en) * | 2002-06-05 | 2007-06-12 | Quantomix Ltd | Methods for SEM inspection of fluid containing samples |
JP2006147430A (en) * | 2004-11-22 | 2006-06-08 | Hokkaido Univ | Electron microscope |
JP2007292702A (en) * | 2006-04-27 | 2007-11-08 | Jeol Ltd | Apparatus, method and system for inspecting samples |
JP2007294365A (en) * | 2006-04-27 | 2007-11-08 | Jeol Ltd | Test piece inspection method, test piece support body, and test piece inspection device as well as test piece inspection system |
JP2008010269A (en) * | 2006-06-28 | 2008-01-17 | Horon:Kk | Low vacuum electronic-optical-system image forming device and low vacuum electronic-optical-system image forming method |
JP5084188B2 (en) * | 2006-07-04 | 2012-11-28 | 日本電子株式会社 | Sample holder, sample inspection method, sample inspection apparatus, and sample inspection system |
JP2008047411A (en) * | 2006-08-15 | 2008-02-28 | Jeol Ltd | Sample retainer, sample inspection method and sample inspection device |
EP1936363A3 (en) * | 2006-12-19 | 2010-12-08 | JEOL Ltd. | Sample inspection apparatus, sample inspection method, and sample inspection system |
JP2009250904A (en) * | 2008-04-10 | 2009-10-29 | Jeol Ltd | Apparatus and method for inspection |
JP5237728B2 (en) * | 2008-08-29 | 2013-07-17 | 日本電子株式会社 | Particle beam equipment |
JP2010230417A (en) * | 2009-03-26 | 2010-10-14 | Jeol Ltd | Inspection apparatus and inspection method for sample |
-
2008
- 2008-03-26 JP JP2008080186A patent/JP5215701B2/en not_active Expired - Fee Related
-
2009
- 2009-03-20 US US12/407,918 patent/US8119994B2/en not_active Expired - Fee Related
- 2009-03-20 EP EP09250792.0A patent/EP2105727B1/en not_active Not-in-force
Also Published As
Publication number | Publication date |
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JP2009238426A (en) | 2009-10-15 |
EP2105727A1 (en) | 2009-09-30 |
US8119994B2 (en) | 2012-02-21 |
US20090242762A1 (en) | 2009-10-01 |
JP5215701B2 (en) | 2013-06-19 |
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